Edited by: Petros Papagerakis, University of Michigan, USA
Reviewed by: Thomas G. H. Diekwisch, Texas A&M University Baylor College of Dentistry, USA; Javier Catón, CEU San Pablo University, Spain
*Correspondence: Domna Dorotheou
This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology
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Tooth eruption, the process by which teeth emerge from within the alveolar bone into the oral cavity, is poorly understood. The post-emergent phase of tooth eruption continues throughout life, in particular, if teeth are not opposed by antagonists. The aim of the present study was to better understand the molecular processes underlying post-emergent tooth eruption. Toward this goal, we removed the crowns of the maxillary molars on one side of the mouth of 14 young rats and examined gene expression patterns in the periodontal ligaments (PDLs) of the ipsilateral and contralateral mandibular molars, 3 and 15 days later. Nine untreated rats served as controls. Expression of six genes, Adamts18, Ostn, P4ha3, Panx3, Pth1r, and Tnmd, was upregulated in unopposed molars relative to molars with antagonists. These genes function in osteoblast differentiation and proliferation, cell adhesion and collagen metabolism. Proliferation of PDL cells also increased following loss of the antagonist teeth. Interestingly, mutations in PTH1R have been linked to defects in the post-emergent phase of tooth eruption in humans. We conclude that post-emergent eruption of unopposed teeth is associated with gene expression patterns conducive to alveolar bone formation and PDL remodeling.
The process, by which teeth emerge from within the alveolar bone into the oral cavity, is referred to as tooth eruption and is divided in two discernible phases: the pre-emergent phase, when the tooth is still in its bony crypt, and the post-emergent phase, which starts when the tooth penetrates the oral mucosa. The mechanisms underlying tooth eruption are still being investigated, yet it already appears that distinct processes operate during the pre- and post-emergent phases (Sarrafpour et al.,
Here, we focused our efforts on the post-emergent phase of tooth eruption. Several studies have shown that this phase is stimulated by loss of the antagonist teeth. One cross-sectional study examined the records of 53 adult individuals having molars without antagonists for at least 10 years and recorded mild overeruption (≤2 mm) in 76% of unopposed molars (Kiliaridis et al.,
Experimental animal models, notably rats, have also been used to study post-emergent eruption of unopposed teeth. In these models, occlusal contacts were eliminated either by extracting the antagonist teeth or by grinding away the tooth crowns. As in humans, the unopposed teeth overerupted and the degree of vertical displacement was more pronounced in younger animals, than in adults (Deporter et al.,
In the animal models, antagonist tooth loss and the resulting post-emergent tooth eruption are associated with changes in the histology of the PDL. First, there is a reduction in the number, diameter and mineral density of Sharpey fibers of the unopposed teeth (Short and Johnson,
In addition to the changes in the Sharpey fibers, post-emergent tooth eruption is associated with changes in the anatomy of the PDL, including narrowing of the periodontal space (Cohn,
Some of the genes possibly involved in the signaling cascades of tooth eruption have been proposed like
In an attempt to gain further insights into the molecular mechanisms underlying post-emergent tooth eruption, we monitored gene expression in the PDL of unopposed molars using a rat model system. We found significant changes in expression only for a handful of genes. Remarkably, mutations in one of these genes, PTH1R, lead to tooth eruption failure in humans.
The experimental protocol was approved by the General Direction of Health, Domain of Animal Experiments, Canton of Geneva, Switzerland. CN: 1080/3807/2.
Twenty-three, 4-week old, male Wistar rats were used in this study: 14 in the experimental group and nine in the control group (Figure - Experimental animals/Unopposed molars (right mandibular molars)/3 or 15 days after crown removal (EU3 and EU15, respectively); - Experimental animals/Opposed molars (left mandibular molars)/3 or 15 days after crown removal (EO3 and EO15, respectively); - Control animals/Right mandibular molars/4-week and 3-day old or 6-week old animals (CR3 and CR15, respectively); - Control animals/Left mandibular molars/4-week and 3-day old or 6-week old animals (CL3 and CL15, respectively).
The unopposed molars in the experimental animals were considered to be hypofunctional; the opposed molars were probably hyperfunctional due to the higher masticatory demands placed on them; while the left and right molars in the control animals should have been subjected to physiological masticatory demands. Since the left and right molars of the control animals could be grouped together, the number of control animals could be lower than the number of experimental animals, thereby minimizing animal suffering (Burden et al.,
Two series of experiments were performed, in order to obtain the material needed for this study.
(a) At the end of the first experiment the first mandibular molars (right and left) were extracted from 15 animals, eight experimental and seven controls. Nine animals, five experimental and four controls were sacrificed 3 days after having cut the maxillary molars and six animals, three experimental and three controls 15 days after having cut the maxillary molars. The PDL was carefully scraped with a scalpel from each root of the molar and RNA extraction was performed. Thus, the RNA from the 30 PDL tissues was used for cDNA microarray and nanostring analysis. (b) In the second experiment we used eight animals, six experimental and two controls for immunohistochemical (IHC) analysis. Four rats, three experimental and one control were sacrificed 3 days and the rest four rats (three experimental and one control), 15 days after having cut the right maxillary molars. Each one of these eight rats received an intraperitoneal single pulse injection of 40 mg/kg BrdU (bromodeoxyuridine) (B5002, Sigma, St. Louis, MO, USA) 1 day before sacrifice. Their mandibles were dissected, fixed, decalcified, dehydrated and embedded in paraffin for further immunohistochemical analysis.
During the experimental period the animals were fed with a soft diet and water
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The extracted RNA was eluted in 30 μl RNase-free sterile water (provided with the kit). Half of the isolated RNA was used for complementary DNA (cDNA) microarray experiments and the other half for Nanostring nCounter Expression Analysis.
Transcriptome profiling was performed using Affymetrix Gene Chip RaGene 2.0 st v1 arrays (Santa Clara, CA, USA), according to the instructions of the manufacturer. Three independent biological replicates were processed per condition, resulting in a total of 24 samples. The quality and quantity of RNA were assessed using a Bioanalyzer (model 2100 with RNA 6000 Nano Chips, Agilent Technologies, Amstelveen, The Netherlands). Two hundred Nano gram of total RNA from each sample was used to prepare double-stranded cDNA, from which biotin-labeled cRNA was synthesized using the BioArray HighYield RNA Transcript Labeling Kit (Enzo). After purification on a QIAGEN RNeasy column, the cRNA was fragmented and hybridized to the arrays. Hybridization, washing and scanning of the arrays were performed according to the manufacturer's instructions. The image data were processed using Affymetrix MAS 5.0 software to generate gene expression data, which were normalized using a robust multi array (RMA) protocol (Bolstad et al.,
Twenty-two RNA samples prepared from PDLs isolated in the first and second series of experiments were processed for Nanostring analysis. Samples corresponding to conditions CR3 and CL3 were considered equivalent, as were samples corresponding to conditions CR15 and CL15. This reduced the number of conditions to six: EO3, EU3, CR3/CL3, EO15, EU15, and CR15/CL15. For each condition, at least three independent, biological replicates were processed.
Expression of 53 genes, 47 experimental and 6 normalization genes was examined using the nCounter system (NanoString). Selection of these genes was based on the transcriptome data obtained by the microarray analysis and on biological insights. The expression of the normalization genes was similar among all groups studied. For each sample, 200 ng of total RNA was hybridized with multiplexed Nanostring probes, as described previously (Geiss et al.,
Sixteen semi-mandibles were dissected for immunohistochemical analysis. Three semi-mandibles were used for each of the following conditions: EO3, EU3, EO15, EU15 and one semi-mandible was used for each of the control conditions: CR3, CL3, CR15, and CL15. The mandibles were fixed in 4% paraformaldehyde (Merck 8.18715, Darmstadt, Germany) for 2 days, decalcified with a solution of 15% EDTA [pH 7.4] and 0.5% paraformaldehyde for 12 weeks, embedded in paraffin, and then sectioned at the frontal plane with a thickness of 3 μm. Paraffin-embedded tissue sections were subjected to deparaffinization and hydration. Endogenous peroxidase activity was then blocked with EnVision Flex Peroxidase-Blocking Reagent (Dako North America, Carpinteria, CA, USA) for 10 min, after which the sections were washed three times with EnVision Flex Wash Buffer (DAko), for 5 min each, followed by denaturation for 20 min in a solution of denaturation (HCL/Triton). Borax (N. 33648, Sigma) treatment was followed for 30 min. The sections were washed three times with EnVision Flex Wash Buffer (DAko), for 5 min each. The sections were then incubated overnight with the primary antibody (anti-BrdU mouse monoclonal antibody; Cat. No. 11170376001, Roche) diluted 1:50 in EnVision Flex Antibody diluent (Daco). The sections were washed three times with EnVision Flex Wash Buffer (DAko), for 5 min each. Incubation with the secondary antibody was followed, (EnVision+ Flex/HRP (K8024, Dako) for 30 min, in room temperature. EnVision Flex DAB (Dako) was used for color development according to the manufacturer's instructions. The sections were counterstained with hematoxylin (No. 1092532500, Merck). Negative control sections were treated in the same manner, except that primary antibody was not added.
For the quantification of BrdU-positive cells, the apical part of the PDL of the first mandibular molars was imaged using a Zeiss (Axio Scan.Z1) microscope in two different areas, vestibular and lingual. Both areas were imaged at 10x and 40x magnification in 8-bit tiff pictures. In order to avoid double-counting of cells, 3 non-consecutive sections with at least 12 μm distance between each section were evaluated from each semi-mandible. A total of 96 images were taken arbitrarily in this region with 40x magnification, 18 from each experimental group (EO3, EU3, EO15, and EU15) and 6 from each control group (CR3, CL3, CR15, and CL15). The total number of cells and the number of positively BrdU-stained cells were measured. The percentage of BrdU-positive cells for each group was calculated and is presented in
The right and left mandibular molars from 23 animals were placed into 8 groups, as described above, based on the presence or absence of antagonist molars, and according to the length of the experimental period, namely 3 or 15 days. In all of the analyses, the results of the right and left molars of the control groups were pooled.
From the 28,407 RefSeq transcripts covered by the Affymetrix Gene Chip 2.0, 700 genes presented more than a 2-fold increase or decrease in expression between the experimental and control groups. From this list, 47 genes were selected for further analysis based on their possible relevance to tooth eruption:
To confirm that the expression of these 47 genes differed between the experimental and control groups, we monitored their expression by a second method, referred to as Nanostring, that detects and counts single mRNA molecules. For this analysis, the genes
Three days after removing the crowns of the maxillary molars in the experimental group,
The third gene examined,
Like
For the last gene examined,
PDL sections were observed and evaluated in the whole length of the root. Proliferation activity was most remarkable in the apical part, mainly in the experimental unopposed and opposed molars for the three- and 15-day samples (Figures
BrdU incorporation was more pronounced in the 3-day than in the 15-day samples. This reflects increased cellular proliferation during the first 3 days following antagonist removal to support eruptive tooth movement, which then decreased with time. In both 3- and 15-day samples, the percentage of BrdU-positive cells was higher in experimental (opposed and unopposed) than in control molars. However, 3-day samples showed significantly greater BrdU incorporation in the PDL of unopposed molars than in opposed molars, whereas 15-day samples showed similar levels for both.
Post-emergent tooth eruption is a multifactorial developmental process involving movement of existing tissues, as well as resorption and formation of new tissues coordinated by a complex set of genetic and metabolic events. In the present study, we have used the model of the unopposed rodent molar to investigate the genetic mechanisms involved in axial tooth movement during post-emergent eruption. Cellular proliferation in the PDL of teeth without antagonists was high and slowed down with time.
The first gene studied,
The observation that overall expression levels of
Tnmd is a transmembrane protein expressed in dense connective tissue such as ligaments and tendons and known to be upregulated in their late developmental stages (Shukunami et al.,
At this point it is worth noting that for the six genes we focused on, the levels of expression were generally lower at the 15 day time point, than at the 3 day time point (Figures
The mechanism of post-emergent tooth eruption remains largely unknown. In our study, we have shown that upon antagonist tooth removal and masticatory force alteration, gene expression of the periodontal ligament changes. In case of experimental unopposed molars, the
In conclusion, we present here a novel implication for
Conceived and designed the experiments: DD, CG, TH, SK. Performed the experiments: DD. Analyzed the data: DD, VF, MB, TH, SK. Wrote the paper: DD, VF, TH, SK.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was funded by the Swiss National Science Foundation (144202) and the Swiss Dental Association (No. 259). The cDNA Microarray and the Nanostring experiments were performed at the iGE3 Genomics Platform of the University of Geneva (
The Supplementary Material for this article can be found online at: